Human IgG1 consists of two Fab (fragment antigen binding) fragments, which comprise the variable regions responsible for antigen recognition, and a constant Fc (fragment crystallizable) domain, which interacts with components of the immune system and mediates immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Carbohydrate structures attached to the conserved N-glycosylation site at asparagine 297 (Asn297, N297) within the CH2 domain of the constant region are mandatory for mediating these effector functions (1-4).
Naturally, the oligosaccharides attached to the Fc domain are predominantly biantennary complex-type structures varying in their content of bisecting GlcNAc (N-acetylglucosamine), terminal galactoses, core fucose and sialic acids (FIG. 1).
Recent studies have shown that modification of the carbohydrate composition strongly affects the antibody-mediated immune effector functions (3-5). A low level of galactosylation positively affects complement activation, while the lack of core fucose results in higher binding affinity to FcγRIIIa and thereby enhances ADCC (5-7). Several approaches have been developed to manipulate the glycosylation profile and to generate therapeutic antibodies with improved biological functions (8-10).
For instance, glycoengineered antibodies produced in mammalian cells overexpressing β(1,4)-N-acetylglucosaminyltransferase (GnT) III and mannosidase (Man) II feature high proportions of bisected, non-fucosylated oligosaccharides and trigger an enhanced ADCC as a result of an up to 50-fold higher affinity for FcγRIIIa (9). However the carbohydrate modifications introduced by overexpression of GnT III, which inhibit the fucosylation reaction, lead to only partially non-fucosylated antibodies. As the Fc domain of an IgG molecule carries two N-linked glycosylation sites, the partial inhibition of the fucosylation reaction can result in a variable distribution of the fucose within an antibody pool. Such an antibody preparation might contain a mixture of molecules carrying one or two fucose residues, while some of them are completely non-fucosylated. Obviously, such different degrees of non-fucosylation influence the overall affinity to FcγRIIIa and result in different biological activity. Therefore, a detailed characterization of such an antibody pool is mandatory.
Since the difference in affinity to FcγRIIIa between fucosylated and non-fucosylated IgG is up to 50-fold, this interaction can be utilized to separate the differently fucosylated species in an antibody pool and characterize them independently.
Existing affinity chromatography matrices used for IgG purification cannot discriminate between different glycosylation patterns within the IgG pool, since the immobilized capture protein specifically binds the protein backbone of the antibody. For instance Protein A and Protein G are binding in the interface between the CH2 and CH3 domain of the Fc region, while other IgG specific proteins such as Protein L are recognizing the constant part of the kappa light chain (11-13).
To enrich proteins carrying specific glycan structures, lectin affinity chromatography has been employed, for example using the Aleuria aurantia lectin (AAL) which binds fucose-containing glycans (14). Alternatively, glycan-targeting antibodies recognizing a specific carbohydrate structure have been used, for example antibodies specific for the Lewis x antigen (15). While these methods may be suitable to enrich glycoproteins carrying a specific carbohydrate, they are of limited use for the enrichment of glycoproteins lacking a specific carbohydrate, such as non-fucosylated antibodies. Moreover, neither of these methods is specific for antibodies and thus would require rigorous purification of an antibody pool prior to its application to the affinity matrix, to avoid contamination by other proteins carrying the targeted glycan structure. Finally, these methods rely on specific lectins or antibodies which may be difficult to obtain, and have not successfully been used for preparative purposes.
Given their greatly increased potency in inducing immune effector function which is of interest for experimental as well as therapeutic purposes, it would be desirable to separate partially or fully non-fucosylated antibodies from fully fucosylated ones present in an antibody pool. The present invention provides a simple and efficient method to achieve such separation.